We propose to continue our efforts to understand, in unusual genetic and molecular detail, how sex is determined in the fruit fly, Drosophila melanogaster. Previous experience has shown how effectively the unique technical features of this system can be exploited to reveal the complexity, diversity, and relatedness of the mechanisms by which developmental processes are controlled in higher organisms. Lessons learned in this model system serve to indicate the kinds of phenomena that are likely to underlie the normal and pathological development of less experimentally tractable organisms like humans and thereby provide insights that will be valuable for improving human health. The rapidity with which primary sex determination mechanisms change, combined with the depth of the understanding of these mechanisms that it is possible to achieve, should eventually lead to important insights into how developmental programs evolve. Methods used in the analysis planned include the most modem molecular approaches being applied to many systems of developmental interest; however, the studies also rely to an unusual degree not just on the more specialized genetic tools available for Drosophila, but also on truly unique genetic tools that have been generated specifically for the study of fruit fly sex determination through nearly two decades of highly focused research in a number of labs. Four areas of investigation are proposed. The first is a continuation of efforts to identify and characterize the individual elements of the polygenic system that is the primary sex determination signal of this organism: the X-chromosome:autosome balance. With the tools generated in this effort, study will begin on the relatedness of this developmental signal in other Drosophila species. The second area deals with understanding the mechanism of sex determination for Drosophila germ cells. It differs in fundamental ways from that which controls the sex of somatic cells. Third are studies aimed at relating gene and protein structure to function for Sex-lethal, the master sex- determination regulatory switch gene that has been the focus of this project since its beginning. Sxl gene products are RNA-binding proteins that control pre-mRNA splicing, including that of the gene's own RNA. It is in this area that the heavy genetic emphasis in this project is perhaps most unusual, a reflection of the special features of the system that allow experimental approaches that would not be feasible in many other situations. Finally, efforts will be made to understand the basis of a new sex-specific lethal mutation that should give new insights into the process of X-chromosome dosage compensation. Ironically, genetic analysis so far has indicated only what this gene is not -- molecular studies are needed to reveal what it is.